The active form of 5-fluorouracil (FU) is its deoxyribonucleotide, FdUMP, which powerfully inhibits the target enzyme, thymidylate synthetase (TS), by formation of a covalent ternary complex between enzyme, inhibitor, and cofactor, 5, 10-methylenetetrahydrofolate. We have now developed new competitive ligand assays for TS and FdUMP that are 100-fold more sensitive than previous assays, and a comparably sensitive assay for dUMP, the substrate of TS. We propose that the FdUMP:dUMP ratio in tumors should be the major determinant of chemotherapeutic response. We propose to determine TS and dUMP before and after a chemotherapeutic dose of FU, and FdUMP and dUMP after the chemotherapeutic dose in 50 mg biopsy specimens obtained from primary and metastatic colorectal, breast, and ovarian carcinomas in 50 patients yearly for three years. These patients will be treated chemotherapeutically with FU and their objective responses will be determined and will be statistically correlated with the FdUMP: dUMP ratios. If a good correlation is found, this will make possible a test for prediction of clinical response to FU. We also propose to develop equally sensitive methods for the quantitative determination of thymidine and the incorporation of FU into RNA. These assays and a newly developed technique for determination of 5, 10-methylenetetrahydrofolate will be added to the clinical study as they become perfected.